Dysregulated metabolism of the late herpes simplex virus 1 transcriptome through the vhs-VP22 axis uncouples virus cytopathic effect and virus production.

Herpes simplex virus 1 (HSV1) expresses its genes in a classical cascade culminating in the production of large amounts of structural proteins to facilitate virus assembly. HSV1 lacking the virus protein VP22 (Δ22) exhibits late translational shutoff, a phenotype that has been attributed to the unrestrained activity of the virion host shutoff (vhs) protein, a virus-encoded endoribonuclease which induces mRNA degradation during infection. We have previously shown that vhs is also involved in regulating the nuclear-cytoplasmic compartmentalisation of the virus transcriptome, and in the absence of VP22 a number of virus transcripts are sequestered in the nucleus late in infection. Here we show that despite expressing minimal amounts of structural proteins and failing to plaque on human fibroblasts, the strain 17 Δ22 virus replicates and spreads as efficiently as Wt virus, but without causing cytopathic effect (CPE). Nonetheless, CPE-causing virus spontaneously appeared on Δ22-infected human fibroblasts, and four viruses isolated in this way had all acquired point mutations in vhs which rescued late protein translation. However, unlike a virus deleted for vhs, these viruses still induced the degradation of both cellular and viral mRNA suggesting that vhs mutation in the absence of VP22 is necessary to overcome a more complex disturbance in mRNA metabolism than mRNA degradation alone. The ultimate outcome of secondary mutations in vhs is therefore the rescue of virus-induced CPE caused by late protein synthesis, and while there is a clear selective pressure on HSV1 to mutate vhs for optimal production of late structural proteins, the purpose of this is over and above that of virus production.

Structural and molecular characteristics of weight-bearing volar skin can be reconstituted by micro skin tissue column grafting.

For patients with lower limb amputations, prostheses are immensely helpful for mobility and the ability to perform job-related or recreational activities. However, the skin covering the amputation stump is typically transposed from adjacent areas of the leg and lacks the weight-bearing capacity that is only found in the specialized skin covering the palms and soles (a.k.a. volar skin). As a result, the skin tissue in direct contact with the prosthesis frequently breaks down, leading to the development of painful sores and other complications that limit, and often preclude, the use of prostheses. Transplanting volar skin onto amputation stumps could be a solution to these problems, but traditional skin transplantation techniques cause substantial morbidity at the donor site, such as pain and scarring, which are especially problematic for volar skin given the critical functional importance of the volar skin areas. We previously developed the technology to collect and engraft full-thickness skin tissue while avoiding long-term donor site morbidity, by harvesting the skin in the form of small (~0.5 mm diameter) cores that we termed "micro skin tissue columns" (MSTCs), so that each donor wound is small enough to heal quickly and without clinically appreciable scarring or other long-term abnormalities. The goal of this study was to establish whether a similar approach could be used to confer the structural and molecular characteristics of volar skin ectopically to other skin areas. In a human-to-mouse xenograft model, we show the long-term persistence of various human plantar MSTC-derived cell types in the murine recipient. Then in an autologous porcine model, we harvested MSTCs from the bottom of the foot and transplanted them onto excision wounds on the animals' trunks. The healing processes at both the donor and graft sites were monitored over 8 weeks, and tissue samples were taken to verify volar-specific characteristics by histology and immunohistochemistry. The volar donor sites were well-tolerated, healed rapidly, and showed no signs of scarring or any other long-term defects. The graft sites were able to maintain volar-specific histologic features and expression of characteristics protein markers, up to the 8-week duration of this study. These results suggest that MSTC grafting could be a practical approach to obtain autologous donor volar skin tissue, confer volar skin characteristics ectopically to nonvolar skin areas, improve the load-bearing capacity of amputation stump skin, and ultimately enhance mobility and quality-of-life for lower limb amputees.

Translational arrest and mRNA decay are independent activities of alphaherpesvirus virion host shutoff proteins.

The herpes simplex virus 1 (HSV1) virion host shutoff (vhs) protein is an endoribonuclease that regulates the translational environment of the infected cell, by inducing the degradation of host mRNA via cellular exonuclease activity. To further understand the relationship between translational shutoff and mRNA decay, we have used ectopic expression to compare HSV1 vhs (vhsH) to its homologues from four other alphaherpesviruses - varicella zoster virus (vhsV), bovine herpesvirus 1 (vhsB), equine herpesvirus 1 (vhsE) and Marek's disease virus (vhsM). Only vhsH, vhsB and vhsE induced degradation of a reporter luciferase mRNA, with poly(A)+ in situ hybridization indicating a global depletion of cytoplasmic poly(A)+ RNA and a concomitant increase in nuclear poly(A)+ RNA and the polyA tail binding protein PABPC1 in cells expressing these variants. By contrast, vhsV and vhsM failed to induce reporter mRNA decay and poly(A)+ depletion, but rather, induced cytoplasmic G3BP1 and poly(A)+ mRNA- containing granules and phosphorylation of the stress response proteins eIF2α and protein kinase R. Intriguingly, regardless of their apparent endoribonuclease activity, all vhs homologues induced an equivalent general blockade to translation as measured by single-cell puromycin incorporation. Taken together, these data suggest that the activities of translational arrest and mRNA decay induced by vhs are separable and we propose that they represent sequential steps of the vhs host interaction pathway.

Herpes Simplex Virus 1 Expressing GFP-Tagged Virion Host Shutoff (vhs) Protein Uncouples the Activities of RNA Degradation and Differential Nuclear Retention of the Virus Transcriptome.

Virion host shutoff (vhs) protein is an endoribonuclease encoded by herpes simplex virus 1 (HSV1). vhs causes several changes to the infected cell environment that favor the translation of late (L) virus proteins: cellular mRNAs are degraded, immediate early (IE) and early (E) viral transcripts are sequestered in the nucleus with polyA binding protein (PABPC1), and dsRNA is degraded to help dampen the PKR-dependent stress response. To further our understanding of the cell biology of vhs, we constructed a virus expressing vhs tagged at its C terminus with GFP. When first expressed, vhs-GFP localized to juxtanuclear clusters, and later it colocalized and interacted with its binding partner VP16, and was packaged into virions. Despite vhs-GFP maintaining activity when expressed in isolation, it failed to degrade mRNA or relocalise PABPC1 during infection, while viral transcript levels were similar to those seen for a vhs knockout virus. PKR phosphorylation was also enhanced in vhs-GFP infected cells, which is in line with a failure to degrade dsRNA. Nonetheless, mRNA FISH revealed that as in Wt but not Dvhs infection, IE and E, but not L transcripts were retained in the nucleus of vhs-GFP infected cells at late times. These results revealed that the vhs-induced nuclear retention of IE and E transcripts was dependent on vhs expression but not on its endoribonuclease activity, uncoupling these two functions of vhs. IMPORTANCE Like many viruses, herpes simplex virus 1 (HSV1) expresses an endoribonuclease, the virion host shutoff (vhs) protein, which regulates the RNA environment of the infected cell and facilitates the classical cascade of virus protein translation. It does this by causing the degradation of some mRNA molecules and the nuclear retention of others. Here, we describe a virus expressing vhs tagged at its C terminus with a green fluorescent protein (GFP) and show that the vhs-GFP fusion protein retains the physical properties of native vhs but does not induce the degradation of mRNA. Nonetheless, vhs-GFP maintains the ability to trap the early virus transcriptome in the nucleus to favor late protein translation, proving for the first time that mRNA degradation is not a prerequisite for vhs effects on the nuclear transcriptome. This virus, therefore, has uncoupled the nuclear retention and degradation activities of vhs, providing a new understanding of vhs during infection.

Association between pelvic pain bothersomeness and pain sensitivity: A community-based cross-sectional study of young adult females in the Raine Study.

OBJECTIVE: Pelvic pain has been associated with augmented nociceptive processing, but large studies controlling for multiple potential confounding factors are lacking. This study investigated the association between pelvic pain bothersomeness and pain sensitivity in young adult women, accounting for potential confounding factors. DESIGN: Cross-sectional study. SETTING: Community-dwelling sample. POPULATION: The Raine Study Gen2-22 year follow-up (n = 475). MAIN OUTCOME MEASURES: The experience of bothersomeness related to pelvic pain was determined from a question in the Urogenital Distress Inventory short form. Pain sensitivity was measured using pressure pain and cold pain thresholds. Potential confounding factors included ethnicity, marital status, highest level of education, income, waist-hip ratio, level of activity, sleep quality, smoking, comorbidity history, C-reactive protein level, musculoskeletal pain experience and psychological distress. RESULTS: Three hundred and sixty-two women (76.2%) reported no pelvic pain bothersomeness, 74 (15.6%) reported mild pelvic pain bothersomeness and 39 (8.2%) reported moderate-severe pelvic pain bothersomeness. After adjusting for marital status (and test site), moderate-severe pelvic pain bothersomeness was associated with a lower pressure pain threshold (i.e. greater pressure pain sensitivity) (coefficient -51.46, 95% CI -98.06 to -4.86, p = 0.030). After adjusting for smoking, moderate-severe pelvic pain bothersomeness was also associated with a higher cold pain threshold (i.e. greater cold pain sensitivity) (coefficient 4.35, 95% CI 0.90-7.79, p = 0.014). CONCLUSIONS: This study suggests augmented nociceptive processing as a contributing factor in pelvic pain bothersomeness for some women. Thorough assessment of women who present clinically with pelvic pain should consider pain sensitivity as a potential contributing factor to their presentation.

Evolutionary Dynamics of Oropouche Virus in South America.

The Amazon basin is home to numerous arthropod-borne viral pathogens that cause febrile disease in humans. Among these, Oropouche orthobunyavirus (OROV) is a relatively understudied member of the genus Orthobunyavirus, family Peribunyaviridae, that causes periodic outbreaks in human populations in Brazil and other South American countries. Although several studies have described the genetic diversity of the virus, the evolutionary processes that shape the OROV genome remain poorly understood. Here, we present a comprehensive study of the genomic dynamics of OROV that encompasses phylogenetic analysis, evolutionary rate estimates, inference of natural selective pressures, recombination and reassortment, and structural analysis of OROV variants. Our study includes all available published sequences, as well as a set of new OROV genome sequences obtained from patients in Ecuador, representing the first set of genomes from this country. Our results show differing evolutionary processes on the three segments that comprise the viral genome. We infer differing times of the most recent common ancestors of the genome segments and propose that this can be explained by cryptic reassortment. We also present the discovery of previously unobserved putative N-linked glycosylation sites, as well as codons that evolve under positive selection on the viral surface proteins, and discuss the potential role of these features in the evolution of OROV through a combined phylogenetic and structural approach.IMPORTANCE The emergence and reemergence of pathogens such as Zika virus, chikungunya virus, and yellow fever virus have drawn attention toward other cocirculating arboviruses in South America. Oropouche virus (OROV) is a poorly studied pathogen responsible for over a dozen outbreaks since the early 1960s and represents a public health burden to countries such as Brazil, Panama, and Peru. OROV is likely underreported since its symptomatology can be easily confounded with other febrile illnesses (e.g., dengue fever and leptospirosis) and point-of-care testing for the virus is still uncommon. With limited data, there is a need to optimize the information currently available. Analysis of OROV genomes can help us understand how the virus circulates in nature and can reveal the evolutionary forces that shape the genetic diversity of the virus, which has implications for molecular diagnostics and the design of potential vaccines.

Isolation of Oropouche Virus from Febrile Patient, Ecuador.

We report identification of an Oropouche virus strain in a febrile patient from Ecuador by using metagenomic sequencing and real-time reverse transcription PCR. Virus was isolated from patient serum by using Vero cells. Phylogenetic analysis of the whole-genome sequence showed the virus to be similar to a strain from Peru.

Molecular Epidemiology and Evolution of European Bat Lyssavirus 2.

Bat rabies cases in Europe are mainly attributed to two lyssaviruses, namely European Bat Lyssavirus 1 (EBLV-1) and European Bat Lyssavirus 2 (EBLV-2). Prior to the death of a bat worker in Finland in 1985, very few bat rabies cases were reported. Enhanced surveillance in the two subsequent years (1986-1987) identified 263 cases (more than a fifth of all reported cases to date). Between 1977 and 2016, 1183 cases of bat rabies were reported, with the vast majority (>97%) being attributed to EBLV-1. In contrast, there have been only 39 suspected cases of EBLV-2, of which 34 have been confirmed by virus typing and presently restricted to just two bat species; Myotis daubentonii and Myotis dasycneme. The limited number of EBLV-2 cases in Europe prompted the establishment of a network of European reference laboratories to collate all available viruses and data. Despite the relatively low number of EBLV-2 cases, a large amount of anomalous data has been published in the scientific literature, which we have here reviewed and clarified. In this review, 29 EBLV-2 full genome sequences have been analysed to further our understanding of the diversity and molecular evolution of EBLV-2 in Europe. Analysis of the 29 complete EBLV-2 genome sequences clearly corroborated geographical relationships with all EBLV-2 sequences clustering at the country level irrespective of the gene studied. Further geographical clustering was also observed at a local level. There are high levels of homogeneity within the EBLV-2 species with nucleotide identities ranging from 95.5-100% and amino acid identities between 98.7% and 100%, despite the widespread distribution of the isolates both geographically and chronologically. The mean substitution rate for EBLV-2 across the five concatenated genes was 1.65 × 10-5, and evolutionary clock analysis confirms the slow evolution of EBLV-2 both between and within countries in Europe. This is further supported by the first detailed EBLV-2 intra-roost genomic analysis whereby a relatively high sequence homogeneity was found across the genomes of three EBLV-2 isolates obtained several years apart (2007, 2008, and 2014) from M. daubentonii at the same site (Stokesay Castle, Shropshire, UK).

Lyssavirus in Indian Flying Foxes, Sri Lanka.

A novel lyssavirus was isolated from brains of Indian flying foxes (Pteropus medius) in Sri Lanka. Phylogenetic analysis of complete virus genome sequences, and geographic location and host species, provides strong evidence that this virus is a putative new lyssavirus species, designated as Gannoruwa bat lyssavirus.

Discovery of hantavirus circulating among Rattus rattus in French Mayotte island, Indian Ocean.

Hantaviruses are emerging zoonotic viruses that cause human diseases. In this study, sera from 642 mammals from La Réunion and Mayotte islands (Indian Ocean) were screened for the presence of hantaviruses by molecular analysis. None of the mammals from La Réunion island was positive, but hantavirus genomic RNA was discovered in 29/160 (18 %) Rattus rattus from Mayotte island. The nucleoprotein coding region was sequenced from the liver and spleen of all positive individuals allowing epidemiological and intra-strain variability analyses. Phylogenetic analysis based on complete coding genomic sequences showed that this Murinae-associated hantavirus is a new variant of Thailand virus. Further studies are needed to investigate hantaviruses in rodent hosts and in Haemorrhagic Fever with Renal Syndrome (HFRS) human cases.

Antigenic and genetic characterization of a divergent African virus, Ikoma lyssavirus.

In 2009, a novel lyssavirus (subsequently named Ikoma lyssavirus, IKOV) was detected in the brain of an African civet (Civettictis civetta) with clinical rabies in the Serengeti National Park of Tanzania. The degree of nucleotide divergence between the genome of IKOV and those of other lyssaviruses predicted antigenic distinction from, and lack of protection provided by, available rabies vaccines. In addition, the index case was considered likely to be an incidental spillover event, and therefore the true reservoir of IKOV remained to be identified. The advent of sensitive molecular techniques has led to a rapid increase in the discovery of novel viruses. Detecting viral sequence alone, however, only allows for prediction of phenotypic characteristics and not their measurement. In the present study we describe the in vitro and in vivo characterization of IKOV, demonstrating that it is (1) pathogenic by peripheral inoculation in an animal model, (2) antigenically distinct from current rabies vaccine strains and (3) poorly neutralized by sera from humans and animals immunized against rabies. In a laboratory mouse model, no protection was elicited by a licensed rabies vaccine. We also investigated the role of bats as reservoirs of IKOV. We found no evidence for infection among 483 individuals of at least 13 bat species sampled across sites in the Serengeti and Southern Kenya.

Diagnosis, management and post-mortem findings of a human case of rabies imported into the United Kingdom from India: a case report.

BACKGROUND: Human rabies infection continues to be a significant public health burden globally, and is occasionally imported to high income settings where the Milwaukee Protocol for intensive care management has recently been employed, with limited success in improving survival. Access to molecular diagnostics, pre- and post-mortem, and documentation of pathophysiological responses while using the Milwaukee protocol, can add useful insights for the future of rabies management. CASE PRESENTATION: A 58-year-old British Asian woman was referred to a regional general hospital in the UK with hydrophobia, anxiety and confusion nine weeks after receiving a dog bite in North West India. Nuchal skin biopsy, saliva, and a skin biopsy from the site of the dog bite wound, taken on the day of admission, all demonstrated the presence of rabies virus RNA. Within 48 hours sequence analysis of viral RNA confirmed the diagnosis and demonstrated that the virus was a strain closely related to canine rabies viruses circulating in South Asia. Her condition deteriorated rapidly with increased agitation and autonomic dysfunction. She was heavily sedated and intubated on the day after admission, treated according to a modified Milwaukee protocol, and remained stable until she developed heart block and profound acidosis and died on the eighth day. Analysis of autopsy samples showed a complete absence of rabies neutralizing antibody in cerebrospinal fluid and serum, and corresponding high levels of virus antigen and nucleic acid in brain and cerebrospinal fluid. Quantitative PCR showed virus was also distributed widely in peripheral tissues despite mild or undetectable histopathological changes. Vagus nerve branches in the heart showed neuritis, a probable Negri body but no demonstrable rabies antigen. CONCLUSION: Rapid molecular diagnosis and strain typing is helpful in the management of human rabies infection. Post-mortem findings such as vagal neuritis highlight clinically important effects on the cardiovascular system which are typical for the clinical course of rabies in humans. Management guided by the Milwaukee protocol is feasible within well-resourced intensive care units, but its role in improving outcome for canine-derived rabies remains theoretical.

Australian healthcare professionals' beliefs and practice behaviours in management of chronic pelvic pain: a cross-sectional survey.

Background Chronic pelvic pain (CPP) is a common and debilitating condition, and sufferers present to healthcare professionals with variable complex symptoms and co-morbidities. This study aimed to investigate the current beliefs and practice behaviours of healthcare professionals towards the management of CPP in Australian females. Methods We distributed an online survey to Australian healthcare professionals. Participants were questioned regarding their beliefs, the importance of various contributing factors and assessment variables, and their management preferences for two CPP vignettes. Demographic information and responses were analysed with descriptive statistics. Results Complete data were obtained and analysed from 446 respondents including gynaecologists (n =75), general practitioners (GPs) (n =184) and physiotherapists (n =187). Most of the respondents were female (88.1%), with male (11.7%) and other (0.2%) making up a smaller representation. Physiotherapists rated themselves higher in understanding mechanisms of CPP (64.7% very good to excellent) compared to gynaecologists (41.3%) and GPs (22.8%). Physiotherapists also reported higher levels of confidence in managing patients with CPP (57.8% quite or extremely confident) compared to 41.3% of gynaecologists and 22.3% of GPs who reported being quite or extremely confident. All three professions rated patient's beliefs (89.8%), nervous system sensitisation (85.7%), stress/anxiety/depression (91.9%), fear avoidance (83.3%), history of sexual/emotional/physical abuse (94.1%) and pelvic floor muscle function (85.0%) as very/extremely important factors in the development of chronic pelvic pain. Most gynaecologists (71.0%) and GPs (70.2%) always referred for pelvic ultrasound during assessment. Physiotherapists assessed goal setting (88.8%) and screened for patients' beliefs (80.9%) more often than gynaecologists (30.4% and 39.1% respectively) and GPs (46.5% and 29.0% respectively). Conclusions All three groups of healthcare professionals demonstrated a good understanding of pain mechanisms and incorporated a biopsychosocial and multidisciplinary approach to management of females with chronic pelvic pain. However, both gynaecologists and GPs were less confident in their understanding of and management of CPP, and less likely to consider patient beliefs and goals. The findings of this online survey may assist in the provision of more targeted education to further improve management of this condition.

A systematic search and narrative review of Aboriginal and Torres Strait Islander women and men pelvic health care: demonstrated need for improvement.

Warning:This article contains terms, descriptions and opinions that may be culturally sensitive for Aboriginal and Torres Strait Islander peoples. BACKGROUND: Pelvic health conditions among Aboriginal and Torres Strait Islander women and men are under-recognised and under-reported despite indication of the significant burden of these conditions. Access to effective management provided in a culturally safe manner appears lacking. The study aims were to: (1) summarise the burden of pelvic health conditions among Aboriginal and Torres Strait Islander peoples; (2) explore barriers and enablers to Aboriginal and Torres Strait Islander peoples accessing pelvic healthcare services; and (3) provide considerations on how to implement culturally safe pelvic healthcare services for Aboriginal and Torres Strait Islander peoples. METHODS: This study involved a systematic literature search informing a narrative review. RESULTS: Fourteen specific articles were identified. Burden related to pelvic health conditions was identified for both women and men, noting high likelihood of under-reporting. Barriers to effective culturally safe treatment included racism, shame and stigma associated with women's and men's business, lack of culturally safe services and geographical distance. Enablers included building trust with local communities, using yarning principles and codesign of pelvic health services with Aboriginal health workers and services. Strategies to improve pelvic health care for Aboriginal and Torres Strait Islander peoples were described at the systems, health service and clinician levels. CONCLUSION: Efforts are needed to improve the diagnosis and management of pelvic health conditions for Aboriginal and Torres Strait Islander peoples. Actions are required to engage with local Aboriginal Community Controlled Aboriginal Community Controlled Health Organisations, professionals overseeing service provision and healthcare providers to address the burden of these conditions among Aboriginal and Torres Strait Islander peoples.

Next generation sequencing of viral RNA genomes.

BACKGROUND: With the advent of Next Generation Sequencing (NGS) technologies, the ability to generate large amounts of sequence data has revolutionized the genomics field. Most RNA viruses have relatively small genomes in comparison to other organisms and as such, would appear to be an obvious success story for the use of NGS technologies. However, due to the relatively low abundance of viral RNA in relation to host RNA, RNA viruses have proved relatively difficult to sequence using NGS technologies. Here we detail a simple, robust methodology, without the use of ultra-centrifugation, filtration or viral enrichment protocols, to prepare RNA from diagnostic clinical tissue samples, cell monolayers and tissue culture supernatant, for subsequent sequencing on the Roche 454 platform. RESULTS: As representative RNA viruses, full genome sequence was successfully obtained from known lyssaviruses belonging to recognized species and a novel lyssavirus species using these protocols and assembling the reads using de novo algorithms. Furthermore, genome sequences were generated from considerably less than 200 ng RNA, indicating that manufacturers' minimum template guidance is conservative. In addition to obtaining genome consensus sequence, a high proportion of SNPs (Single Nucleotide Polymorphisms) were identified in the majority of samples analyzed. CONCLUSIONS: The approaches reported clearly facilitate successful full genome lyssavirus sequencing and can be universally applied to discovering and obtaining consensus genome sequences of RNA viruses from a variety of sources.

Complete genome sequence of Ikoma lyssavirus.

Lyssaviruses (family Rhabdoviridae) constitute one of the most important groups of viral zoonoses globally. All lyssaviruses cause the disease rabies, an acute progressive encephalitis for which, once symptoms occur, there is no effective cure. Currently available vaccines are highly protective against the predominantly circulating lyssavirus species. Using next-generation sequencing technologies, we have obtained the whole-genome sequence for a novel lyssavirus, Ikoma lyssavirus (IKOV), isolated from an African civet in Tanzania displaying clinical signs of rabies. Genetically, this virus is the most divergent within the genus Lyssavirus. Characterization of the genome will help to improve our understanding of lyssavirus diversity and enable investigation into vaccine-induced immunity and protection.

Pathogenesis of Rift Valley Fever Virus in a BALB/c Mouse Model Is Affected by Virus Culture Conditions and Sex of the Animals.

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen causing disease in livestock and humans. Whilst initially restricted to the African continent, recent spread to the Arabian Peninsula has highlighted the likelihood of entry into new regions. Due to the absence of a regulatory-approved human vaccine, work is ongoing to develop and assess countermeasures. As such, small animal models play a pivotal role in providing information on disease pathogenesis and elucidating which intervention strategies confer protection. To develop and establish the BALB/c mouse model, we challenged mice with RVFV grown from two separate cell lines: one derived from mosquitoes (C6/36) and the other mammalian derived (Vero E6). Following infection, we assessed the clinical course of disease progression at days 1 and 3 post-challenge and evaluated viral tropism and immune analytes. The results demonstrated that RVFV infection was affected by the cell line used to propagate the challenge virus, with those grown in insect cells resulting in a more rapid disease progression. The lowest dose that caused uniform severe disease remained the same across both virus preparations. In addition, to demonstrate reproducibility, the lowest dose was used for a subsequent infection study using male and female animals. The results further demonstrated that male mice succumbed to infection more rapidly than their female counterparts. Our results establish an RVFV mouse model and key parameters that affect the course of disease progression in BALB/c mice.

A distinct subset of human NK cells expressing HLA-DR expand in response to IL-2 and can aid immune responses to BCG.

Subsets of NK cells can have distinct functions. Here, we report that >25% of human peripheral blood NK cells express HLA-DR after culture with IL-2. This can be driven by an expansion of a small subset of NK cells expressing HLA-DR, in contrast to previous assumptions that HLA-DR is upregulated on previously negative cells. HLA-DR-expressing NK cells showed enhanced degranulation to susceptible target cells and expressed chemokine receptor CXCR3, which facilitated their enrichment following exposure to CXCL11/I-TAC. Suggesting HLA-DR-expressing NK cells have an important role in an immune response, stimulation of PBMCs with Mycobacterium bovis BCG (BCG) triggered expansion of this subset. Importantly, the magnitude of an individual's NK cell IFN-γ response triggered by BCG was associated with the initial frequency of HLA-DR-expressing NK cells in PBMCs. More directly indicating the importance of HLA-DR-expressing NK cells, enriching the frequency of this subset in PBMCs substantially augmented the IFN-γ response to BCG. Thus, HLA-DR expression marks a distinct subset of NK cells, present at low frequency in circulating blood but readily expanded by IL-2, that can play an important role during immune responses to BCG.

The CXCL16 A181V mutation selectively inhibits monocyte adhesion to CXCR6 but is not associated with human coronary heart disease.

OBJECTIVE: The chemokine CXCL16 serves as a scavenger receptor for oxidized low-density lipoprotein and as an adhesion molecule and chemoattractant for cells expressing the receptor CXCR6. A commonly occurring CXCL16 allele has been described containing 2 nonsynonymous single-nucleotide polymorphisms in complete linkage disequilibrium, although the effects on CXCL16 function are unknown. Here, we examined the effect of the single-nucleotide polymorphisms on CXCL16 function and assessed the association of the mutant allele with coronary heart disease (CHD). METHODS AND RESULTS: Both wild-type and mutant T123V181-CXCL16 were readily expressed in vitro and were similarly functional in assays of oxidized low-density lipoprotein scavenging and chemotaxis. However, unlike wild-type CXCL16, T123V181-CXCL16 was unable to promote adhesion of CXCR6(+) cells. Findings were confirmed ex vivo, with monocytes from donors homozygous for the T123V181 allele unable to facilitate adhesion of CXCR6 transfectants. In the London Life Sciences Prospective Population cohort (n = 2797), we found that the T123V181 allele was not associated with protection or susceptibility to CHD (adjusted odds ratio, 1.01; 95% CI, 0.95 to 1.10; P = 0.74). CONCLUSIONS: CXCL16-mediated cell adhesion plays at best a modest role in CHD, and the scavenging and chemotactic properties of the chemokine are more likely to be more important in disease pathogenesis.